Prior studies.sup.1 have shown that remarkably specific irreversible enzyme inhibitors can result from compounds bearing potential reactive groupings which are unmasked at the active site by the target enzyme. This specificity resides in the generation of the alkylating agent by the target enzyme at the active site as a result of the enzyme's normal catalytic process. The process is exemplified by the enzymatic conversion of an acetylenic compound to an allene which can alkylate an active site amino acid residue. The first such example was provided by Bloch.sup.2 who showed that the acetylenic analog of a normal substrate for .beta.-hydroxydecanoyl thioester dehydrase is converted by the enzyme to the corresponding conjugated allenic thioester with rapid alkylation of an active site histidine residue. This approach has been applied to the inhibition of monoamine oxidase.sup.3 and .gamma.-cystathionase..sup.4 FNT (1) Cf. R. R. Rando, Science, 185, 320 (1974). (2) K. Bloch, Acc. Chem. Res. 2, 193 (1969); K. Endo, G. M. Helmkamp, and K. Bloch, J. Biol. Chem. 245, 4293 (1970); M. Morisaka and K. Bloch, Bioorg. Chem., 1 188 (1971). (3) R. R. Rando, J. Am. Chem. Soc., 95, 4438 (1973); R. R. Rando and J. De Mairena, Biochem. Pharmacol., 23, 463 (1974); R. C. Hevey, J. Babson, A. L. Maycock, and R. H. Abeles, J. Am. Chem. Soc., 95, 6125 (1973). (4) R. H. Abeles and C. T. Walsh, J. Am. Chem. Soc. 95, 6124 (1973).
The enzyme .DELTA..sup.5 -3-ketosteroid isomerase.sup.5 (EC 5.3.3.1) from Pseudomonas testosteroni converts C.sub.19 and C.sub.21 .DELTA..sup.5 -3-ketosteroids to the corresponding .DELTA..sup.4 -3-ketosteroids. The proposed mechanism.sup.5,6 involves removal of the axial 4.beta.-hydrogen with concomitant enolization to give a .DELTA..sup.3,5 -dienol, followed by ketonization with axial reprotonation at C-6. The hydrogen transfer from C-4 to C-6 is intramolecular. See Scheme 1 below: ##STR1## The indicated reaction, when carried out by mammalian .DELTA..sup.5 -3-ketosteroid isomerases is a key step in the biosynthesis of steroid hormones. FNT (5) P. Talalay and A. M. Benson, Enzymes, 6, 591 (1972). (6) S. K. Malhotra and H. J. Ringold, J. Am. Chem. Soc., 87, 3228 (1965).